Project Summary/Abstract Pulmonary fibrosis is a common disorder without effective pharmacological treatment. Fibrosis is a pathological tissue response characterized by extracellular deposition of collagen-rich matrix that disrupts normal alveolar architecture preventing effective gas exchange. The metabolism of collagen, the main component of fibrotic tissue, is a dynamic process with the balance between collagen production and degradation determining tissue architecture. While there has been considerable research directed toward identifying the pathways responsible for collagen production in pulmonary fibrosis, the pathways responsible for removal of collagen accumulated in areas of fibrosis are poorly understood. Whether therapies aimed at increasing collagen uptake will benefit patients with established fibrotic disease is unknown. We have recently found the glycoprotein Milk Fat Globule Epidermal Growth Factor like 8 (Mfge8) binds and targets collagen for intracellular uptake and degradation by macrophages. Mice deficient in Mfge8 develop an exaggerated fibrotic response to bleomycin injury due to an in vivo defect in collagen degradation. The overall objective of this proposal is to investigate the role of macrophage-mediated collagen phagocytosis in limiting the severity of pulmonary fibrosis. We will pursue this objective through 3 specific aims. In the proposed experiments, we will determine which domains of Mfge8 are critical for collagen binding and collagen uptake. We will map the molecular pathway by which Mfge8-bound collagen is taken up by cells. We will examine the role of macrophages in Mfge8-dependent and independent remodeling of pulmonary fibrosis in vivo through a series of macrophage depletion experiments. In this fashion, we will investigate the pathways by which macrophages degrade collagen. A better understanding of these pathways will provide novel targets for therapies aimed at treating established fibrotic disease. Our specific aims are: The first aim is to determine which domains of Mfge8 are critical for collagen binding and uptake. We will create a series of Mfge8 constructs with truncated and/or mutated sequences and evaluating their function in in vitro and in vivo assays of collagen binding and uptake. We will use cells obtained from Mfge8-/- and wild type mice. The second aim is to determine the role EGFR receptor family and macropinocytosis in Mfge8- dependent collagen uptake. We will accomplish this aim through a series of in vitro experiments delineating the mechanism by which Mfge8-bound collagen activates EGFR and how EGFR activation induces collagen uptake through macropinocytosis. The final aim is to determine the role of pulmonary macrophages in collagen resorption in vivo. We will use both pharmacological approaches (Clodronate and anti-F4/80 antibody) and transgenic approaches of macrophage depletion (Mafia mice and CD11b-DTR mice) to determine the in vivo role of macrophage-mediated collagen degradation in pulmonary fibrosis. We will deplete macrophages after fibrosis induced by bleomycin treatment is established in the lung and then evaluate the effect of macrophage depletion on the normal remodeling of collagen-rich scar. We will also determine the cellular source of Mfge8 important for lung remodeling through a series of bone marrow ablation and immune reconstitution experiments. These studies have the potential to identify novel pathways and mediators of collagen uptake that can be targeted to treat established pulmonary fibrosis.